Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase

1 Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes the endogenous nitric oxide synthase inhibitors N(G)-monomethyl-L-arginine and N(G),N(G)-dimethy-L-arginine to citrulline, was identified by Western blotting in rat and human tissue homogenates. 2 S-2-amino-4(3-methylguanidino)butanoic acid (4124W) inhibited the metabolism of [(14)C]-N(G)-monomethyl-L-arginine to [(14)C]-citrulline by rat liver homogenates (IC(50) 416+/-66 mu M; n=9), human cultured endothelial cells (IC(50) 250+/-34 mu M; n=9) and isolated purified dimethylarginine dimethylaminohydrolase. 3 Addition of 4124W to culture medium increased the accumulation of endogenously-generated N(G),N(G)-dimethy-L-arginine in the supernatant of human cultured endothelial cells from 3.1+/-0.3 to 5+/-0.7 mu M (n=15; P< 0.005). 4 4124W (1 mu M-1 mM) had no direct effect on endothelial nitric oxide synthase activity but caused endothelium-dependent contraction of rat aortic rings (1 mM 4124W increased tone by 81.5+/-9.6% of that caused by phenylephrine 100 nM). This effect was reversed by L-arginine (100 mu M) 4124W reversed endothelium-dependent relaxation of human saphenous vein (19.2+/-6.7% reversal of bradykinin-induced relaxation at 1 mM 4124W). 5 These data suggest that inhibition of dimethylarginine dimethylaminohydrolase increases the intracellular concentration of N(G),N(G)-dimethyl-L-arginine sufficiently to inhibit nitric oxide synthesis. Inhibiting the activity of DDAH may provide an alternative mechanism for inhibition of nitric oxide synthases and changes in the activity of DDAH could contribute to pathophysiological alterations in NO generation.